2. Academic Validation
  3. Development and validation of a fluorescence polarization-based assay for USP7: From probe design to inhibitor evaluation

Development and validation of a fluorescence polarization-based assay for USP7: From probe design to inhibitor evaluation

  • Eur J Med Chem. 2026 Aug 5:312:118857. doi: 10.1016/j.ejmech.2026.118857.
Siji Chen 1,  Mingchen Wang 2,  Yasi Zeng 3,  Xinyuan Li 4,  Hui Zhong 4,  Yiling Liu 3,  Yunsu Tao 3,  Xu Yang 3,  Cheng Luo 5,  Shijie Chen 6,  Huan Xiong 7
Affiliations

Affiliations

  • 1 State Key Laboratory of Discovery and Utilization of Functional Components in Traditional Chinese Medicine, School of Pharmaceutical Sciences, Guizhou Medical University, Guiyang, 550004, China; Zhongshan Institute for Drug Discovery, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Zhongshan, 528400, China.
  • 2 School of Pharmaceutical Science and Technology, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou, 310024, China.
  • 3 Innovation Center for AI and Drug Discovery, School of Pharmacy, East China Normal University, Shanghai, 200062, China.
  • 4 Zhongshan Institute for Drug Discovery, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Zhongshan, 528400, China; Guangzhou University of Chinese Medicine, Guangdong, 510006, China.
  • 5 State Key Laboratory of Discovery and Utilization of Functional Components in Traditional Chinese Medicine, School of Pharmaceutical Sciences, Guizhou Medical University, Guiyang, 550004, China; Zhongshan Institute for Drug Discovery, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Zhongshan, 528400, China; School of Pharmaceutical Science and Technology, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou, 310024, China; Innovation Center for AI and Drug Discovery, School of Pharmacy, East China Normal University, Shanghai, 200062, China; Guangzhou University of Chinese Medicine, Guangdong, 510006, China. Electronic address: [email protected].
  • 6 Innovation Center for AI and Drug Discovery, School of Pharmacy, East China Normal University, Shanghai, 200062, China. Electronic address: [email protected].
  • 7 Zhongshan Institute for Drug Discovery, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Zhongshan, 528400, China. Electronic address: [email protected].
PMID: 42001546 DOI: 10.1016/j.ejmech.2026.118857
Abstract

Ubiquitin-Specific Protease 7 (USP7) is a key member of the deubiquitinating enzyme family. It is abnormally overexpressed in various malignancies, including breast Cancer, chronic lymphocytic leukemia, and prostate Cancer. By regulating pathways such as the p53-MDM2 signaling axis, USP7 promotes tumorigenesis and progression, making it a highly promising therapeutic target for Anticancer treatment. Although multiple USP7 inhibitors have been reported, existing screening and evaluation assays exhibit limitations: the ubiquitin-phospholipase A2 (Ub-PLA2) assay frequently produces false-positive results, while the ubiquitin-rhodamine (Ub-Rho) assay is susceptible to interference from compound autofluorescence. To address this challenge, we developed a fluorescence polarization (FP) assay. This employs a rationally designed strategy that exhibits excellent characteristics, making it a simple-to-operate and cost-effective method, suitable for the evaluation of compound bioactivity against USP7. To further validate the practicality and reliability of this FP assay, we conducted a structure-based drug design campaign involving two rounds of systematic structural optimization, yielding 51 novel derivatives featuring pyrazolo[4,3-d]pyrimidine and piperidol scaffolds. Following FP evaluation and Ub-Rho enzyme activity validation, we performed a comprehensive structure-activity relationship (SAR) analysis. Ultimately, in vitro cellular assays identified three compounds (LC-U7-44, LC-U7-48, and LC-U7-50) that exhibit potent USP7 inhibitory activity alongside favorable cellular anti-proliferative effects. Overall, the established FP assay in this study closes a methodological gap in the evaluation of USP7 inhibitors, and the detailed SAR analysis provides a foundation for the further development of potent USP7 inhibitors.

Keywords

Anti-cancer agents; Fluorescence polarization (FP); Inhibitor evaluation; Structure-based drug design; Ubiquitin-specific protease 7 (USP7).

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